Our understanding of how HIV interacts with the immune system in humans remains limited, and this has impeded development of strategies to elicit effective protective immunity and to reduce pathogenic effects of the virus. While the T cell response to HIV has been studied extensively in humans, the role of cells involved in the innate response to HIV and other retroviruses has received relatively little attention. Dendritic cells (DC) play a major role in detecting and initiating the response to pathogens, thus linking the innate immune response to adaptive immunity. They serve as early response sensors of microbial infection and also as specialized antigen presenting cells in the induction of T cell responses (Steinman and Hemmi, 2006) (Takeuchi and Akira, 2007a). They are also likely to play a major role in the innate response to HIV: there is a dense network of DC at mucosal surfaces where HIV enters the organism and replicates extensively early after transmission (Niess et al., 2005; Veazey and Lackner, 2003), and the function of these cells in immune responses to pathogenic and commensal microorganisms has only recently begun to be explored.
In recent years, it has been recognized that there are multiple types of DC, including myeloid DC and plasmacytoid DC, which differ in both pathogen recognition mechanisms and in effector functions (Colonna et al., 2004; Cao and Liu, 2007). At mucosal surfaces, particularly in the intestinal lamina propria and in gut-draining lymph nodes, there are myeloid DC with distinct phenotypes that can elicit either inflammatory or tolerogenic responses in T cells. These distinct DC subsets have been studied most extensively in the mouse, in which they can be subdivided into CX3CR1hiCD103− cells that produce abundant IL-6 and IL-23 and CX3CR1loCD103+ cells that produce retinoic acid and induce the differentiation of Foxp3+ regulatory T cells (Coombes et al., 2007; Sun et al., 2007; Mucida et al., 2007). The inflammatory DC have recently also been described in human mesenteric lymph nodes (Jaensson et al., 2008; Moris et al., 2006). The balance of the different types of DC is likely to have a key role in host protection and also in immune system homeostasis, preventing excessive inflammation, particularly in response to normally harmless commensal microorganisms. It should be noted, however that the functions proposed for the diverse subsets of DC are based largely on in vitro studies, and have not been confirmed in mice or humans.
Dendritic Cells and HIV Infection
Several aspects of HIV interactions with DC have been studied. It was noted early by Steinman and colleagues that DC potentiate infection of co-cultured T cells with HIV-1 without themselves being infected (Cameron et al., 1992b). DC have been shown to mediate HIV capture in endocytic-like compartments, which can lead either to infectious transfer to T cells or to degradation and possibly antigen presentation on MHC molecules (Wu and KewalRamani, 2006). DC express HIV receptors (CD4 and CCR5), which allows HIV entry into the cytoplasm to be detected (using assays such as Vpr-Blam) (Cavrois et al., 2006). However, dendritic cells are strikingly resistant to productive HIV infection. In vivo, while macrophages are clearly infected, there is no clear indication that mucosal dendritic cells are infected with HIV (Cameron et al., 1992a). In vitro, monocyte-derived dendritic cells (MDDC, the most extensively characterized model of dendritic cells) are also extremely resistant to HIV infection. In fact, using standard MOI's, infection cannot be detected with single-round HIV-1-based vectors encoding GFP or luciferase (Boggiano et al., 2007). Several publications have reported p24 accumulation in MDDC culture supernatants after 2 weeks of infection, but it is unclear whether p24 originated from MDDC or other cells or whether it was derived from true infection events (i.e. integrative) (Turville et al., 2004). It has been reported that low levels of HIV-1 transduction of MDDC can be achieved when high MOIs, i.e. at least 10, are used (Goujon et al., 2003; Negre et al., 2000). The resistance, which thus appears saturable by high amounts of virus, is reminiscent of classical Fv1 restriction of MLV (Duran-Troise et al., 1977; Goff, 1996). Using a GFP reporter virus, we have indeed confirmed that extremely high MOI of virus obtained by ultracentrifugation can lead to detectable events of infection by flow cytometry (Manel and Littman, unpublished). It is thus likely that MDDC possess strong native restriction to HIV-1 infection.
Recent progress has contributed to a better understanding of DC restriction to HIV-1 replication. It was found that while HIV-1 vectors were unable to infect DC, SIVmac vectors were fully capable of doing so (Mangeot et al., 2000). SIVmac and HIV-2 encode Vpx, a protein that is absent in HIV-1 and that is essential for effective infection of DC, but not T cells, by Sly. Remarkably, infection of MDDC with Vpx-harboring SIVmac virus-like particles (SIVmac VLP) rendered the cells susceptible to HIV infection (Goujon et al., 2006). Recently, Vpx-containing VLPs were also found to enhance production of full-length cDNA with feline and murine retroviruses (Goujon et al., 2007). Thus, Vpx appears to inactivate one or more host cell factors that restrict replication of HIV-1 and other retroviruses in MDDC (and, to a lesser extent, in macrophages). Using cell-fusion assays, it has further been shown that the Vpx-sensitive factor is dominant in endowing cells with resistance to infection with HIV-1 (Sharova et al., 2008). Replication of HIV-1 in dendritic cells is abrogated early after viral entry, before reverse transcription is completed (and thus resembles restriction by TRIM5a). Vpx has been shown to interact with DCAF1, which, in turn, binds to the DDB1/CUL4 E3 ligase complex. A functional role for this interaction is suggested by the finding that siRNA-mediated knockdown of DCAF1 in primary macrophages resulted in substantially reduced transduction efficiency by SIVmac239 (Srivastava et al., 2008). However, another recent study has questioned the requirement for DCAF1 association with Vpx in Vpx-dependent infection of differentiated THP-1 cells and, to a lesser extent, MDDC (Goujon et al., 2008). Moreover, the DCAF1 complex is broadly expressed and is not restricted to monocyte lineage cells. It is therefore highly likely that one or more factors whose expression is restricted to DC, and not T cells, is responsible for the early block of HIV replication in DC.
Although DCs are largely resistant to productive infection with HIV-1, they have the remarkable ability to enhance in trans infection of activated CD4+ T cells. In vitro studies have shown that, when HIV is incubated at low MOI with T cells, inclusion of MDDC results in much more efficient infection of the T cells (Cameron et al., 1992b; Geijtenbeek et al., 2000). The mechanism for trans-infection has been a subject of some controversy. Initial studies indicated that the C-type lectin DC-SIGN was required for DC-mediated enhancement (Geijtenbeek et al., 2000; Kwon et al., 2002), but recently we and others have shown that this is not an essential requirement and there are likely multiple DC surface molecules that interact with the HIV envelope to mediate viral internalization and enhanced infection of T cells (Boggiano et al., 2007; Gummuluru et al., 2003). We had shown that HIV that is transmitted from DC to T cells is taken up by DC into a protease-resistant non-lysosomal compartment (Kwon et al., 2002). Another recent report argued that only surface-bound protease-sensitive and soluble CD4-sensitive virus is transmitted from DC to T cells (Cavrois et al., 2007). In that report, however, high virus titers were employed, and the phenomenon resembled that observed when RAJI cells transfected with DC-SIGN were used in place of the DC. It has been suggested that virus is taken up into compartments that remain contiguous with the plasma membrane, allowing access to inhibitory molecules such as soluble CD4. This interpretation is consistent with the more recent finding that HIV is taken up into a novel compartment adjacent to the plasma membrane (Yu et al., 2008). It has been proposed that virus may be carried by lipid rafts on the plasma membrane towards a synapse with T cells (Cavrois et al., 2008). What is clear at this point is that DCs are endowed with a specialized means to enhance the infectivity of T cells with HIV, but the mechanism for this process remains obscure. Moreover, the significance of this DC function in vivo has yet to be explored. Thus, the contribution of mucosal DC to the explosive rapid replication of HIV-1 and SIV in intestinal lamina propria T cells shortly after infection of humans and non-human primates, respectively, has yet to be examined.
Innate Anti-Viral Response Mechanisms
During the past decade, there has been renewed appreciation of the importance of innate immunity in protection from microbial infections. Innate immune responses, which are evolutionarily much more ancient than adaptive responses found in vertebrates (Hoffmann and Reichhart, 2002), can directly limit the replication of infectious agents and also act indirectly by activating B and T cell responses (Medzhitov, 2007). Mechanisms of innate immunity include recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs), lectin family receptors, cytoplasmic NOD/NALP-like receptors (NLRs), and cytoplasmic helicase domain proteins such as RIG-I and MDA5 (RLHs) (Takeuchi and Akira, 2007b) (Takeda and Akira, 2005) (Stetson and Medzhitov, 2006). Despite substantial progress in understanding innate responses to bacteria, fungi, and many viruses, there is little known of vertebrate innate immune responses that limit replication of retroviruses. It has been proposed that the single-strand RNA-detecting TLR7 is involved in responses to HIV, but evidence is limited to the ability of viral RNA to elicit a TLR7-mediated response, and there is little evidence that HIV particles can trigger such a response on their own (Beignon et al., 2005). Cytoplasmic helicase domain proteins RIG-I and MDA5 have essential roles in antiviral innate responses in a variety of cells other than plasmacytoid DC. These molecules have been shown to be important in responses against several RNA viruses. RIG-I is activated by non-capped RNAs that have a 5′ terminal triphosphate and polyuridine or polyriboadenine motifs in the 3′ UTRs (Hornung et al., 2006; Saito et al., 2008). The role of the RLHs in anti-retroviral innate immunity has not been reported, and the genomic retroviral messenger RNA is 5′ capped. Recognition of cytoplasmic DNA may also be expected to constitute a mechanism of innate immunity against retroviruses. A sensor for cytoplasmic DNA, DAI, was described recently, but it is likely that there are multiple such recognition molecules (Takaoka et al., 2007). The potential importance of this and other cytoplasmic DNA sensors is highlighted by recent studies on the cytoplasmic 3′-5′ ssDNA exonuclease Trex1. Defects in the gene encoding Trex1 have been shown to be linked to Aicardi-Goutieres Syndrome, an inflammatory disease of the nervous system, and mice with deficiency of Trex1 have autoimmune myocarditis. It has now been shown that, in the absence of Trex1, there is an increase in DNA retroelements in the cytoplasm, activation of IRF3, and increased production of type I interferon (Stetson et al., 2008). The autoimmune myocarditis is mediated by the adaptive immune response, since Trex1-deficient mice that also lack RAG2 have no disease. The retroelements likely activate innate immune recognition machinery, including DAI. These recent results suggest that Trex1, and related members in the family of cytoplasmic exonucleases, degrade cytoplasmic DNA that is generated by reverse transcription of endogenous retroelements and thus prevent abnormal activation of innate immune responses and production of interferons and other effector cytokines. In addition to preventing inflammation, Trex1 may also protect cells from reverse transcribed DNA generated from infections with retroviruses. Thus, a better understanding of how b-interferon is induced in the absence of Trex1 (or related exonucleases expressed in tissues other than myocardium) may provide insight into the mechanism by which host cells sense retrovirus components in the cytoplasm and transmit the information to the adaptive immune system.
Some insight into innate immune system requirements for activation of an adaptive anti-retroviral immune response has come from studies in mice of infection with the Friend virus (FV), a complex retrovirus that is widely used as a model for understanding anti-retroviral immune responses and mechanisms of persistent infection (Hasenkrug and Chesebro, 1997; Miyazawa et al., 2008). FV consists of a mixture of a helper virus and a defective virus that encodes gp55, which interacts with erythropoietin receptor and drives proliferation of erythroblasts. In C57BL/6 mice, infection is followed by peak viral titers in the spleen at 7 dpi and resolution to a low level persistent viremia. During the acute phase of infection, the virus is controlled by both T cell and B cell responses that follow an early peak in type I interferon production (Gerlach et al., 2006). We have investigated the roles played by dendritic cells and by the TLR signaling pathway in control of FV infection in mice. Both DC and the Myd88 signaling pathway, employed by all TLRs except for TLR3, were required for generation of an antiviral antibody response (Browne and Littman, 2009). It remains unclear whether the Myd88 signal is required in DC and whether a known TLR interacts with a feature of the retroviral infection that serves as a PAMP (pathogen-associated molecular pattern) to set the innate response into motion. It is also not known whether TLR signaling is important in activation of an adaptive immune response following infection with HIV.
Relationship Between Infection of Dc with HIV-1 and Activation of an Innate Immune Response
Although it has not yet been feasible to perform studies in humans to investigate the in vivo role for DCs following HIV infection, it is highly likely that these cells perform key functions in mounting an innate immune response to limit viral replication. A better understanding of how innate responses are initiated and how they function after HIV infection is critical for achieving better vaccine strategies. Because DCs are the most potent cell type for initiation of innate immune responses, particularly for activating antigen-specific T cells, the means by which HIV interacts with DCs is likely to be crucial for setting the quality and quantity of innate immunity. A key question that arises is whether exposure of DCs to HIV-1 under conditions of productive infection versus non-productive enhancement of T cell infection results in different outcomes in terms of activation of DC innate functions. A few attempts have been made to address related questions in various systems (Boasso et al., 2008; Fantuzzi et al., 2004; Goujon et al., 2006; Granelli-Piperno et al., 2004; Smed-Sorensen et al., 2005; Smed-Sorensen et al., 2004). However, in the absence of Vpx, the rare infection events observed with HIV-1 did not appear to activate DC, but the efficiency of infection was so low (1%) that it is risky to interpret this result conclusively (Granelli-Piperno et al., 2004). In the presence of Vpx, transduction of minimal lentiviral vector devoid of the expression of viral proteins did not seem to induce MDDC activation (Goujon et al., 2006). It would be useful to determine whether resistance of DCs to infection with HIV-1 is beneficial to the host, potentially limiting spread of the virus, or detrimental, due to limitation in the level of innate immune system activation that can result in stronger anti-viral B and T cell responses.
The interactions of HIV and SIV with the host innate immune system may explain, at least in part, the puzzling differences between individuals (and different non-human primate species) in their ability to contain the infection and avoid immune deficiency. Characterization of host genes and proteins involved in regulating myeloid cell infection and the host innate anti-viral immune response may provide clues that can be applied to the future investigation of how individual variation can explain host susceptibility to HIV disease.
Thus, while the innate response to several viruses is known, the response to HIV-1 has remained elusive. A better understanding of innate immune response to HIV-1 and a knowledge of the molecules or markers involved would be useful, particularly in designing, generating, and developing HIV vaccines or in stimulating responses to HIV in infected or at risk individuals. Accordingly, it would be desirable to identify molecules or targets involved in innate response to HIV and HIV-like pathogens and to provide novel molecules and methods for improving HIV vaccine strategies and it is toward the achievement of these objectives that the present invention is directed.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.